The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. We presented the sequence of a new HLA-B*08 variant, HLA-B*0804, found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B*51 in JH and HLA-B*4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B*08 variant, HLA-B*0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B*0804 differed from HLA-B*0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. OBJECTIVE: To define a new B locus variant. RESULTS This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles. FUTURE DIRECTIONS This paper demonstrates the use of our HLA-B typing technique. KEY WORDS cross-reactivity, DGGE, HLA-B*0804, PCR-SSP, sequence, typing